Edrington T C, Yeagle P L, Gretzula Cheryl L, Boesze-Battaglia K
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Biochemistry. 2007 Mar 27;46(12):3862-71. doi: 10.1021/bi061999r. Epub 2007 Feb 27.
Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.
外周蛋白/视网膜变性慢（p/rds）是跨膜4（TMF4）超家族的一种整合膜蛋白，其多功能的C末端结构域在视杆细胞外段（ROS）盘状结构的更新和维持中起着关键作用。在此，我们报道钙结合蛋白钙调蛋白（CaM）能与p/rds的C末端结构域结合。荧光光谱显示CaM与对应于p/rds C末端结构域的多肽存在Ca2+依赖性结合。CaM滴定后多肽的荧光各向异性得出解离常数（KD）为320±150 nM。荧光实验结果通过GST下拉分析得到证实，其中GST-p/rds C末端结构域融合蛋白能以钙依赖方式下拉CaM。此外，分子建模和序列预测表明，CaM结合结构域位于p/rds功能热点区域，即E314和G329残基之间。肽竞争研究以及一个假定的Ca2+/CaM结合位点被打乱的GST-p/rds C末端结构域构建体证实了这一预测。该GST多肽不与Ca2+/CaM结合。这一假定的钙调蛋白结构域在人、小鼠、大鼠和牛的p/rds中高度保守。最后，在模型膜研究中，Ca2+/CaM的结合抑制了ROS盘状膜与ROS质膜之间的融合以及p/rds C末端结构域诱导的融合。这些结果为p/rds功能调节提供了一种新机制。
